This proposal aims to elucidate patterns of inheritance within a single species of ciliates to address three specific hypotheses. These hypotheses focus on variation in the content of (1) somatic (macronuclear) genomes and (2) germline (micronuclear) genomes, and on (3) the possible role of epigenetics in shaping the generation of somatic genomes following sexual conjugation. These hypotheses are based on preliminary data from two isolates (one from the USA and one from Poland) of a single morphospecies, Chilodonella uncinata. Although the isolates contain identical ribosomal DNAs and share some identical protein coding sequences, they also show complex patterns of inheritance. Duplicated copies of genes are differentially inherited in macronuclei following conjugation and cross between lines alter patterns of inheritance of at least one gene, actin. The resulting data from this project will shed light on patterns of inheritance within the ciliate Chilodonella uncinata, and also on epigenetic processes that are likely analogous, and perhaps even homologous, to events in other eukaryotes. [unreadable] [unreadable] To address these hypotheses, the proposed work will combine generation of clone libraries, qPCR, variant-specific PCR, and fluorescent microscopy. These methods are either already in use in the PIs laboratory or, in the case of fluorescent microscopy, currently under development. [unreadable] [unreadable] Finally, the proposed work will enable the PI to continue to involve students (both undergraduate and graduate) in her research program. The PI has a demonstrated record in recruiting students from traditionally underrepresented groups, and will continue with these efforts as part of the proposed work. The proposed work on ciliates is relevant to public health in that resulting discoveries will yield insights into developmentally-regulated genome processing in eukaryotic cells (cells with nuclei). The type of genome processing to be studied in this proposal is analogous to changes in genomes in human immune cells and in some types of cancers. Studies of microbial eukaryotes, including ciliates, have already led to discovery of numerous genome features that have broad relevance to humans, including the presence of telomeres on the end of chromosomes and the existence of self-splicing RNA. [unreadable] [unreadable] [unreadable]